Cytotoxic T-lymphocyte-inducing immunogens for prevention, treatment, and diagnosis of cancer

ABSTRACT

The present invention relates to compositions and methods for the prevention, treatment, and diagnosis of cancer, especially carcinomas, such as ovarian carcinoma. The invention discloses peptides, polypeptides, and polynucleotides that can be used to stimulate a CTL response against cancer.

This application claims priority of U.S. Provisional Application No.60/251,022, filed Dec. 4, 2000, and No. 60/256,824, filed 20 Dec. 2000,the disclosures of which are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates generally to the field of immunogens whosestructures incorporate polypeptides comprising epitopic peptides derivedfrom proteins expressed by cancer cells and to uses of said immunogensin eliciting cytotoxic T lymphocyte (CTL) responses for the diagnosis,prevention and treatment of cancer, preferably carcinoma, mostpreferably ovarian carcinoma.

BACKGROUND OF THE INVENTION

The mammalian immune system has evolved a variety of mechanisms toprotect the host from cancerous cells, an important component of thisresponse being mediated by cells referred to as T cells. Cytotoxic Tlymphocytes (CTLs) are specialized T cells that function primarily byrecognizing and killing cancerous cells or infected cells, but also bysecreting soluble molecules referred to as cytokines that can mediate avariety of effects on the immune system.

Evidence suggests that immunotherapy designed to stimulate atumor-specific CTL response would be effective in controlling cancer.For example, it has been shown that human CTLs recognize sarcomas(Slovin, S. F. et al., J. Immunol., 137:3042–3048, (1987)), renal cellcarcinomas (Schendel, D. J. et al., J. Immunol., 151:4209–4220, (1993)),colorectal carcinomas (Jacob, L. et al., Int. J. Cancer, 71:325–332,(1997)), ovarian carcinomas (loannides, C. G. et al., J. Immunol.,146:1700–1707, (1991)) (Peoples, G. E. et al., Surgery, 114:227–234,(1993)), pancreatic carcinomas (Peiper, M. et al., Eur. J. Immunol.,27:1115–1123, (1997); Wolfel, T. et al., Int. J. Cancer, 54:636–644,(1993)), squamous tumors of the head and neck (Yasumura, S. et al.,Cancer Res., 53:1461–1468, (1993)), and squamous carcinomas of the lung(Slingluff, C. L. Jr et al., Cancer Res., 54:2731–2737, (1994); Yoshino,I. et al., Cancer Res., 54:3387–3390, (1994)). The largest number ofreports of human tumor-reactive CTLs have concerned cancers (Boon, T. etal., Ann. Rev. Immunol., 12:337–365, (1994)). The ability oftumor-specific CTLs to mediate tumor regression, in both human(Rosenberg, S. A. et al., N. Engl. J. Med., 319:1676–1680, (1988)) andanimal models (Celluzzi, C. M. et al., J. Exp. Med., 183:283–287,(1996); Mayordomo, J. I. et al., Nat. Med., 1:1297–1302, (1995);Zitvogel, L. et al., J. Exp. Med., 183:87–97, (1996)), suggests thatmethods directed at increasing CTL activity would likely have abeneficial effect with respect to tumor treatment.

In order for CTLs to kill or secrete cytokines in response to a cancercell, the CTL must first recognize that cell as being cancerous. Thisprocess involves the interaction of the T cell receptor, located on thesurface of the CTL, with what is generically referred to as anMHC-peptide complex which is located on the surface of the cancerouscell. MHC (major histocompatibility-complex)-encoded molecules have beensubdivided into two types, and are referred to as class I and class IIMHC-encoded molecules.

In the human immune system, MHC molecules are referred to as humanleukocyte antigens (HLA). Within the MHC, located on chromosome six, arethree different genetic loci that encode for class I MHC molecules. MHCmolecules encoded at these loci are referred to as HLA-A, HLA-B, andHLA-C. The genes that can be encoded at each of these loci are extremelypolymorphic, and thus, different individuals within the populationexpress different class I MHC molecules on the surface of their cells.HLA-A1, HLA-A2, HLA-A3, HLA-B7, and HLA-B8 are examples of differentclass I MHC molecules that can be expressed from these loci. The presentdisclosure involves peptides that are associated with the HLA-A1/A11 orHLA-A2 molecules, and with the gene and protein that gives rise to thesepeptides.

The peptides that associate with the MHC molecules can either be derivedfrom proteins made within the cell, in which case they typicallyassociate with class I MHC molecules (Rock, K. L. and Golde, U., Ann.Rev. Immunol., 17:739–779, (1999)) or they can be derived from proteinsthat are acquired from outside of the cell, in which case they typicallyassociate with class II MHC molecules (Watts, C., Ann. Rev. Immunol.,15:821–850, (1997)). Peptides that evoke a cancer-specific CTL responsemost typically associate with class I MHC molecules. The peptides thatassociate with a class I MHC molecule are typically nine amino acids inlength, but can vary from a minimum length of eight amino acids to amaximum of fourteen amino acids in length. A class I MHC molecule withits bound peptide, or a class II MHC molecule with its bound peptide, isreferred to as an MHC-peptide complex.

The process by which intact proteins are degraded into peptides isreferred to as antigen processing. Two major pathways of antigenprocessing occur within cells (Rock, K. L. and Golde, U., Ann. Rev.Immunol., 17:739–779, (1999); Watts, C., Ann. Rev. Immunol., 15:821–850,(1997)). One pathway, which is largely restricted to cells that areantigen presenting cells such as dendritic cells, macrophages, and Bcells, degrades proteins that are typically phagocytosed or endocytosedinto the cell. Peptides derived in this pathway typically bind to classII MHC molecules. A second pathway of antigen processing is present inessentially all cells of the body. This second pathway primarilydegrades proteins that are made within the cells, and the peptidesderived from this pathway primarily bind to class I MHC molecules. It isthe peptides from this second pathway of antigen processing that arereferred to herein. Antigen processing by this latter pathway involvespolypeptide synthesis and proteolysis in the cytoplasm. The peptidesproduced are then transported into the endoplasmic reticulum of thecell, associate with newly synthesized class I MHC molecules, and theresulting MHC-peptide complexes are then transported to the cellsurface. Peptides derived from membrane and secreted proteins have alsobeen identified. In some cases these peptides correspond to the signalsequence of the proteins that are cleaved from the protein by the signalpeptidase. In other cases, it is thought that some fraction of themembrane and secreted proteins are transported from the endoplasmicreticulum into the cytoplasm where processing subsequently occurs.

Once bound to the class I MHC molecule and displayed on the surface of acell, the peptides are recognized by antigen-specific receptors on CTLs.Mere expression of the class I MHC molecule itself is insufficient totrigger the CTL to kill the target cell if the antigenic peptide is notbound to the class I MHC molecule. Several methods have been developedto identify the peptides recognized by CTL, each method relying on theability of a CTL to recognize and kill only those cells expressing theappropriate class I MHC molecule with the peptide bound to it(Rosenberg, S. A., Immunity, 10:281–287, (1999)). Such peptides can bederived from a non-self source, such as a pathogen (for example,following the infection of a cell by a bacterium or a virus) or from aself-derived protein within a cell, such as a cancerous cell. Examplesof sources of self-derived proteins in cancerous cells have beenreviewed (Gilboa, E., Immunity, 11:263–270, (1999); Rosenberg, S. A.,Immunity, 10:281–287, (1999)) and include: (i) mutated genes; (ii)aberrantly expressed genes such as an alternative open reading frame orthrough an intron-exon boundary; (iii) normal genes that are selectivelyexpressed in only the tumor and the testis; and (iv) normaldifferentiation genes that are expressed in the tumor and the normalcellular counterpart.

Four different methodologies have typically been used for identifyingthe peptides that are recognized by CTLs. These are: (i) the geneticmethod; (2) motif analysis; (3) SErological analysis of REcombinant cDNAexpression libraries (SEREX™); and (iv) the analytical chemistryapproach or the Direct Identification of Relevant Epitopes for ClinicalTherapeutics (DIRECT™).

The genetic method is an approach in which progressively smaller subsetsof cDNA libraries from tumor cells are transfected into cells thatexpress the appropriate MHC molecule but not the tumor-specific epitope.The molecular clones encoding T cell epitopes are identified by theirability to reconstitute tumor specific T cell recognition of transfectedcells. The exact T cell epitope is then identified by a combination ofmolecular subcloning and the use of synthetic peptides based on thepredicted amino acid sequence. Such methods, however, are susceptible toinadvertent identification of cross-reacting peptides, and are notcapable of identifying important post-translational modifications.

Motif analysis involves scanning a protein for peptides containing knownclass I MHC binding motifs, followed by synthesis and assay of thepredicted peptides for their ability to be recognized by tumor-specificCTL. This approach requires prior knowledge of the protein from whichthe peptides are derived. This approach is also greatly hampered by thefact that not all of the predicted peptide epitopes are presented on thesurface of a cell (Yewdell, J. W. and Bennink, J. R., Ann. Rev.Immunol., 17:51–88, (1999)), thus additional experimentation is requiredto determine which of the predicted epitopes is useful.

The SEREX™ approach relies on using antibodies in the serum of cancerpatients to screen cDNA expression libraries for a clone that expressesa protein recognized by the antibody. This methodology presumes that anantibody response will necessarily have developed in the presence of a Tcell response, and thus, the identified clone is a good candidate toencode a protein that can be recognized by T cells.

DIRECT™ involves a combination of cellular immunology and massspectrometry. This approach involves the actual identification of CTLepitopes by sequencing the naturally occurring peptides associated withclass I MHC molecules. In this approach, cells are first lysed in adetergent solution, the peptides associated with the class I MHCmolecules are purified, and the peptides fractionated by highperformance liquid chromatography (HPLC). The peptides are then used toreconstitute recognition by tumor-specific CTLs on a non-tumor cellexpressing the appropriate MHC molecules. Sequencing is readilyperformed by tandem mass spectrometry (Henderson, R. A. et al., Proc.Natl. Acad. Sci. U.S.A, 90:10275–10279, (1993); Hogan, K. T. et al.,Cancer Res., 58:5144–5150, (1998); Hunt, D. F. et al., Science,255:1261–1263, (1992); Slingluff, C. L. Jr et al., J. Immunol.,150:2955–2963, (1993)).

Immunization with cancer-derived, class I MHC-encodedmolecule-associated peptides, or with a precursor polypeptide or proteinthat contains the peptide, or with a gene that encodes a polypeptide orprotein containing the peptide, are forms of immunotherapy that can beemployed in the treatment of cancer. These forms of immunotherapyrequire that immunogens be identified so that they can be formulatedinto an appropriate vaccine. Although a variety of cancer-derivedantigens have been identified (Rosenberg, S. A., Immunity, 10:281–287,(1999)), not all of these are appropriate for broad-based immunotherapyas the expression of some peptides is limited to the tumor derived froma specific patient. Furthermore, the number of class I MHC moleculesfrom which tumor-derived peptides have been discovered is largelyrestricted to HLA-A2. Thus, it would be useful to identify additionalpeptides that complex with class I MHC molecules other than HLA-A2. Suchpeptides would be particularly useful in the treatment of cancerpatients who do not express the HLA-A2 molecule. The HLA-A1/A11 antigensof the present invention (SEQ ID NO: 17–20) would be examples. It isalso particularly useful to identify antigenic peptides that are derivedfrom different parent proteins, even if the derived peptides associatewith the same class I MHC molecule. Because an active immune responsecan result in the outgrowth of tumor cells that have lost the expressionof a particular precursor protein for a given antigenic peptide, it isadvantageous to stimulate an immune response against peptides derivedfrom more than one parent protein, as the chances of the tumor celllosing the expression of both proteins is the multiple of the chances oflosing each of the individual proteins.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to Immunogens comprising polypeptides withamino acid sequences comprising epitopic sequences selected from thesequences of SEQ ID NO: 1–20 and which immunogens facilitate a cytotoxicT lymphocyte (CTL)-mediated immune response against cancers. The presentinvention also relates to nucleic acid molecules that encode for thepolypeptides and/or the full length proteins from which the polypeptidesare derived, of such immunogens, and which can also be used tofacilitate an immune response against cancer.

The present invention provides compositions comprising the immunogendescribed herein, and polynucleotides that direct the synthesis of suchpolypeptides, whereby the oligopeptides and polypeptides of suchimmunogens are capable of inducing a CTL response against cellsexpressing a protein comprising an epitopic sequence of SEQ ID NO: 1–16presented in association with HLA-A2 and SEQ ID NO: 17–20 presented inassociation with HLA-AI/AII. The cells are usually cancer cells,preferably carcinoma cells, most preferably ovarian carcinomasexpressing such proteins.

The present invention further relates to polynucleotides comprising thegene coding for a polypeptide of the immunogens disclosed herein.

The present invention also provides methods that comprise contacting alymphocyte, especially a CTL, with an immunogen of the invention underconditions that induce a CTL response against a tumor cell, and morespecifically against a cancer cell. The methods may involve contactingthe CTL with the immunogenic peptide in vivo, in which case thepeptides, polypeptides, and polynucleotides of the invention are used asvaccines, and will be delivered as a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and the immunogen,typically along with an adjuvant or one or more cytokines.

Alternatively, the immunogens of the present invention can be used toinduce a CTL response in vitro. The generated CTL can then be introducedinto a patient with cancer, more specifically cancer, colorectalcarcinoma, ovarian carcinoma, lung carcinoma, or prostate carcinoma.Alternatively, the ability to generate CTL in vitro could serve as adiagnostic for cancer generally, including colorectal carcinoma, ovariancarcinoma, lung carcinoma, or prostate carcinoma.

DEFINITIONS

As used herein and except as noted otherwise, all terms are defined asgiven below.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are typically 9 amino acids in length, but can be as short as 8amino acids in length, and as long as 14 amino acids in length.

The term “oligopeptide” is used herein to designate a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.The length of the oligopeptide is not critical to the invention as longas the correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 14 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to proteinmolecules of longer than about 30 residues in length.

A peptide, oligopeptide, protein, or polynucleotide coding for such amolecule is “immunogenic” (and thus an “immunogen” within the presentinvention) if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a CTL-mediated response. Thus, an “immunogen”would be a molecule that is capable of inducing an immune response, andin the case of the present invention, a molecule capable of inducing aCTL response.

A T cell “epitope” is a short peptide molecule that binds to a class Ior II MHC molecule and that is subsequently recognized by a T cell. Tcell epitopes that bind to class I MHC molecules are typically 8–14amino acids in length, and most typically 9 amino acids in length. Tcell epitopes that bind to class II MHC molecules are typically 12–20amino acids in length. In the case of epitopes that bind to class II MHCmolecules, the same T cell epitope may share a common core segment, butdiffer in the length of the carboxy- and amino-terminal flankingsequences due to the fact that ends of the peptide molecule are notburied in the structure of the class II MHC molecule peptide-bindingcleft as they are in the class I MHC molecule peptide-binding cleft.

As used herein, reference to a DNA sequence includes both singlestranded and double stranded DNA. Thus, the specific sequence, unlessthe context indicates otherwise, refers to the single strand DNA of suchsequence, the duplex of such sequence with its complement (doublestranded DNA) and the complement of such sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene. The coding region can be from anormal, mutated or altered gene, or can even be from a DNA sequence, orgene, wholly synthesized in the laboratory using methods well known tothose of skill in the art of DNA synthesis.

The term “nucleotide sequence” refers to a heteropolymer ofdeoxyribonucleotides. The nucleotide sequence encoding for a particularpeptide, oligopeptide, or polypeptide may be naturally occurring or theymay be synthetically constructed. Generally, DNA segments encoding thepeptides, polypeptides, and proteins of this invention are assembledfrom cDNA fragments and short oligonucleotide linkers, or from a seriesof oligonucleotides, to provide a synthetic gene which is capable ofbeing expressed in a recombinant transcriptional unit comprisingregulatory elements derived from a microbial or viral operon.

The term “expression product” means that polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment,” when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnontranslated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that is pairedwith one strand of DNA and provides a free 3′OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “open reading frame (ORF)” means a series of triplets codingfor amino acids without any termination codons and is a sequence(potentially) translatable into protein.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, the claimed polypeptide which has a purity of preferably0.001%, or at least 0.01% or 0.1%; and even desirably 1% by weight orgreater is expressly contemplated.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form.” As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform.

The term “active fragment” means a fragment that generates an immuneresponse (i.e., has immunogenic activity) when administered, alone oroptionally with a suitable adjuvant, to an animal, such as a mammal, forexample, a rabbit or a mouse, and also including a human, such immuneresponse taking the form of stimulating a CTL response within therecipient animal, such as a human. Alternatively, the “active fragment”may also be used to induce a CTL response in vitro.

As used herein, the terms “portion,” “segment,” and “fragment,” whenused in relation to polypeptides, refer to a continuous sequence ofresidues, such as amino acid residues, which sequence forms a subset ofa larger sequence. For example, if a polypeptide were subjected totreatment with any of the common endopeptidases, such as trypsin orchymotrypsin, the oligopeptides resulting from such treatment wouldrepresent portions, segments or fragments of the starting polypeptide.This means that any such fragment will necessarily contain as part ofits amino acid sequence a segment, fragment or portion, that issubstantially identical, if not exactly identical, to a sequence of SEQID NOs: 1–4. When used in relation to polynucleotides, such terms referto the products produced by treatment of said polynucleotides with anyof the common endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical,” when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The Percent Identity isthen determined according to the following formula:Percent Identity=100 [1−(C/R)]wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence wherein (i) each base oramino acid in the Reference Sequence that does not have a correspondingaligned base or amino acid in the Compared Sequence and (ii) each gap inthe Reference Sequence and (iii) each aligned base or amino acid in theReference Sequence that is different from an aligned base or amino acidin the Compared Sequence, constitutes a difference; and R is the numberof bases or amino acids in the Reference Sequence over the length of thealignment with the Compared Sequence with any gap created in theReference Sequence also being counted as a base or amino acid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated Percent Identity is less than the specified PercentIdentity.

DETAILED SUMMARY OF THE INVENTION

The present invention relates generally to immunogens and immunogeniccompositions, and methods of use therefor, for the prevention,treatment, and diagnosis of cancer, especially carcinomas, includingovarian carcinomas. Disclosed according to the invention are immunogenscomprising proteins or polypeptides whose amino acid sequences comprisesone or more epitopic oligopeptides with sequences selected from thegroup SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19 and 20. In addition, the invention further relates topolynucleotides that can be used to stimulate a CTL response againstcancer, and more specifically carcinoma, especially ovarian carcinomas.

In accordance with the present invention there are disclosed specificoligopeptide sequences with amino acid sequences shown in SEQ ID NO:1–20, which represent epitopic peptides (i.e. immunogenic oligopeptidesequences) of at least about 8 amino acids in length, preferably about 9amino acids in length (i.e., nonapeptides), and no longer than about 10amino acids in length and present as part of a larger structure, such asa polypeptide or full length protein. Proteins already known asimmunogens in the art, such as MAGE 4 and MFG-E8, are specificallyexcluded from the present invention, especially where the epitopicpeptide is one of SEQ ID NO: 17, 18, 19 and 20.

The polypeptides forming the immunogens of the present invention haveamino acid sequences that comprise at least one stretch, possibly two,three, four, or more stretches of about 8 to 10 residues in length andwhich stretches differ in amino acid sequence from the sequences of SEQID NO: 1–20 by no more than about 1 amino acid residue, preferably aconservative amino acid residue, especially amino acids of the samegeneral chemical character, such as where they are hydrophobic aminoacids.

Said polypeptides can be of any desired length so long as they haveimmunogenic activity in that they are able, under a given set ofdesirable conditions, to elicit in vitro or in vivo the activation ofcytotoxic T lymphocytes (CTLs) (i.e., a CTL response) against apresentation of a cancer specific protein, especially a carcinoma orsarcoma specific protein, most especially MAGE D, MAGE 4, MFG-E8 orhuman retinoblastoma-like protein, especially when such proteins arepresented along with MHC-1 proteins, such as where said proteins arepresented in vitro or in vivo by an antigen presenting cell (APC). Theproteins and polypeptides forming the immunogens of the presentinvention can be naturally occurring or may be synthesized chemically.

The present invention is also directed to an isolated polypeptide,especially one having immunogenic activity, the sequence of whichcomprises within it one or more stretches comprising any 2 or more ofthe sequences of SEQ ID NO: 1–20 and in any relative quantities andwherein said sequences may differ by one amino acid residues from thesequences of SEQ ID NO: 1–20 in any given stretch of 8 to 10 amino acidresidues. Thus, within the present invention, an by way of anon-limiting example only, such polypeptide may contain as part of itsamino acid sequence, nonapeptide fragments having up to 8 amino acidsidentical to a sequence of SEQ ID NO: 1–4 such that the polypeptidecomprises, in a specific embodiment, 2 segments with at least 8 residuesidentical to SEQ ID NO: 1 and one segment with at least 8 residuesidentical to SEQ ID NO: 3. In other embodiments, other combinations andpermutations of the epitopic sequences disclosed herein may be part ofan immunogen of the present invention or of such a polypeptide so longas any such polypeptide comprises at least 2 such epitopes, whether suchepitopes are different or the same. Thus, in a specific embodiment, apolypeptide of the present invention may comprise 2 copies of thesequence of SEQ ID NO: 2 at some point or points within its length. Ofcourse, any combinations and permutations of the epitopes disclosedherein, as long as they are present at least two in number in suchpolypeptides, are expressly contemplated.

All of the epitopic peptides of SEQ ID NO: 1–20 are derived fromproteins expressed by cancer cells and sequences and were identifiedthrough the method of Automated High Through-put Sequencing (HTPS).

Epitopic nonapeptide of SEQ ID NO: 1 (KIMDQVQQA) is derived from thehuman APC gene expressed by an ovarian carcinoma cell line, OVCAR3.While having been used in a vector for a vaccine [See: Chada et al, U.S.Pat. No. 5,693,522], this protein has not been described as an immunogenbut has been linked to other types of cancer [See: Spirio et al, Nat.Genetics, 20, 385–8 (1998)].

The epitopic decapeptide of SEQ ID NO: 2 (RLQEDPPAGV) is derived fromthe human ubiquitin conjugating enzyme (HHR6A) and was expressed by theOVCAR3 ovarian carcinoma cell line. The sequence was identified usingHTPS. [See: Lal et al, U.S. Pat. No. 6,013,702 for a description of thisprotein; Pati et al, Mol. Cell Biol., 19:5001–13 (1999) for adescription of the functions of the enzyme in targeting cyclicAMP-induced transcription].

The epitopic nonapeptide of SEQ ID NO: 3 (KLDVGNAEV) is derived fromaccessory protein BAP 31 and was expressed by the OVCAR3 ovariancarcinoma cell line. [See: Granville et al, FEBS Lett. 437:5–10 (1998);Annaert et al, J. Cell Biol., 139:1397–1410 (1997); Li et al, Eur. J.Biochem., 238:631–638 (1996); Ng et al, J. Cell Biol., 139:327–338(1997); Ng and Shore, J. Biol. Chem., 273:3140–43 (1998)] The sequencewas identified using HTPS.

The epitopic nonapeptide of SEQ ID NO: 4 (FLYDDNQRV) is derived fromTopoisomerase II and was expressed by the OVCAR3 ovarian carcinoma cellline. [See: Kim et al, Anticancer Res., 19:5393–8 (1999); Fortune andOsheroff, Prog. Nuc. Acid Res. Mol. Biol., 64:221–253 (2000); Willmanand Holden, Prostate, 42:280–286 (2000); Cowell et al, Exp. Cell Res.,255:86–94 (2000); Lage et al, Br. J. Cancer, 82:488–91 (2000)] Thesequence was identified using HTPS.

The epitopic nonapeptide of SEQ ID NO: 5 (ALMEQQHYV) is derived fromIntegrin beta-8 subunit precursor and was expressed by the OVCAR3ovarian carcinoma cell line. [See:Nishimura et al, J. Biol. Chem.,269:28708–15 (1994); Moyle et al, J. Biol. Chem., 266:19650–8 (1991)]The sequence was identified using HTPS.

The epitopic nonapeptide of SEQ ID NO: 6 (YLMDTSGKV) is derived fromreplication protein A and was expressed by the SKOV3 A2 ovariancarcinoma cell line. The sequence was identified using HTPS.

The epitopic nonapeptide of SEQ ID NO: 7 (ILDDIGHGV) is derived from AbIbinding protein 3 and was expressed by the SKOV3 A2 and OVCAR3 ovariancarcinoma cell lines. The sequence was identified using HTPS.

The epitopic nonapeptide of SEQ ID NO: 8 (LLDRFLATV) is derived fromCyclin protein and was expressed by the SKOV3 A2 ovarian carcinoma cellline. The sequence was identified using HTPS.

The epitopic eicosapeptide of SEQ ID NO: 9 (LLIDDKGDIKL) is derived fromprotein encoded by human CDC2 gene involved in control of the celilcycle and was expressed by the SKOV3 A2 ovarian carcinoma cell line. Thesequence was identified using HTPS.

The epitopic decapeptide of SEQ ID NO: 10 (RLYPWGVVEV) is derived fromthe protein encoded by human messenger RNA for the KIAA0158 gene and wasexpressed by the SKOV3 A2 ovarian carcinoma cell line. The sequence wasidentified using HTPS.

The epitopic nonapeptide of SEQ ID NO: 11 (KLQELNYNL) is derived from asignal transducer and activator protein and was expressed by the SKOV3A2 ovarian carcinoma cell line. The sequence was identified using HTPS.

The epitopic nonapeptide of SEQ ID NO: 12 (ILIEHLYGL) is derived fromLDL receptor related protein and was expressed by the SKOV3 A2 ovariancarcinoma cell line. The sequence was identified using HTPS.

The epitopic nonapeptide of SEQ ID NO: 13 (YLIELIDRV) is derived fromTNF-alpha converting enzyme and was expressed by the OVCAR3 ovariancarcinoma cell line. The sequence was identified using HTPS.

The epitopic nonapeptide of SEQ ID NO: 14 (NLMEQPIKV) is derived fromPlakoglobin protein and was expressed by the OVCAR3 ovarian carcinomacell line. The sequence was identified using HTPS.

The epitopic decapeptide of SEQ ID NO: 15 (FLAEDALNTV) is derived fromEDDR1 gene for receptor tyrosine kinase and was expressed by the OVCAR3ovarian carcinoma cell line. The sequence was identified using HTPS.

The epitopic nonapeptide of SEQ ID NO: 16 (TLLNVIKSV) is derived frominositol triphosphate receptor type II and was expressed by the OVCAR3and SKOV3 ovarian carcinoma cell lines. The sequence was identifiedusing HTPS.

Epitopic nonapeptide of SEQ ID NO: 17 (MLKDIIKEY) is derived from theprotein MAGE D, expressed by an ovarian carcinoma cell line. MAGE D isalso known as hepatocellular carcinoma associated protein—JCL-1, asSnt-1 breast cancer associated protein, and as MAGE-like protein. [See:Lucas et al, Cancer Res., 59:4100–4103 (1999)].

The epitopic nonapeptide of SEQ ID NO: 18 (TSYVKVLEH) is derived fromthe protein MAGE 4 and was expressed by an ovarian carcinoma cell linedifferent from that for the other epitopic peptides disclosed herein.The sequence was identified using HTPS. [See: Boon-Falleur et al, U.S.Pat. No. 5,695,994 for a description of a MAGE 3 protein; Chen et al,Liver, 19:110–114 (1999) for a description of the expression of MAGEgenes in human carcinomas].

The epitopic nonapeptide of SEQ ID NO: 19 (HEYLKAFKV) is derived fromthe protein MFG-E8 protein and was expressed by an ovarian carcinomacell line different from that for the other epitopic peptides disclosedherein. This protein is also known as the milk fat globule EGF(epidermal growth factor) Factor 8 protein and as BA46 and lactadherin.[See: Taylor et al, DNA Cell Biol., 16:861–869 (1997)]

The epitopic nonapeptide of SEQ ID NO: 20 (IYIKQIKTF) is derived fromthe human retinoblastoma-like protein (p130), also called E1A-associated130 k protein or E1A-associated cyclin-binding 130 k protein, and wasexpressed by an ovarian carcinoma cell line different from that for theother epitopic peptides disclosed herein. [See: Claudio et al, CancerRes., 60:372–382 (2000)]

Oligopeptides as disclosed herein may themselves be prepared by methodswell known to those skilled in the art. (Grant, G. A., SyntheticPeptides: A User's Guide, 1992, W.H. Freeman and Company, New York;Coligan, J. E. et al, Current Protocols in Protein Science, 1999, JohnWiley & Sons, Inc., New York).

Besides the sequences of SEQ ID NOs:1–20, the proteins and polypeptidesforming the immunogens of the present invention may also comprise one ormore other immunogenic amino acid stretches known to be associated withcancer, and more specifically with carcinomas and melanomas, includingcolorectal carcinoma, ovarian carcinoma, lung carcinoma, or prostatecarcinoma, and which may stimulate a CTL response whereby theimmunogenic peptides associate with HLA-A1/A11, or HLA-A2, or anotherclass I MHC (i.e., MHC-1) molecule.

The immunogens of the present invention can be in the form of acomposition of one or more of the different immunogens and wherein eachimmunogen is present in any desired relative abundance. Suchcompositions can be homogeneous or heterogeneous with respect to theindividual immunogenic peptide components present therein, having onlyone or more than one of such peptides.

The oligopeptides and polypeptides useful in practicing the presentinvention may be derived by fractionation of naturally occurringproteins by methods such as protease treatment, or they may be producedby recombinant or synthetic methodologies that are well known and clearto the skilled artisan (Ausubel, F. M. et al, Current Protocols inMolecular Biology, 1999, John Wiley & Sons, Inc., New York; Coligan, J.E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons,Inc., New York; Molecular Cloning: A Laboratory Manual, 1989, ColdSpring Harbor Laboratory Press, Cold Spring Harbor). The polypeptide maycomprise a recombinant or synthetic polypeptide that necessarilycomprises at least one of SEQ ID NOs:1–20 which sequences may also bepresent in multiple copies. Thus, oligopeptides and polypeptides of thepresent invention may have one, two, three, or more such immunogenicpeptides within the amino acid sequence of said oligopeptides andpolypeptides, and said immunogenic peptides, or epitopes, may be thesame or may be different, or may have any number of such sequenceswherein some of them are identical to each other in amino acid sequencewhile others within the same polypeptide sequence are different fromeach other and said epitopic sequences may occur in any order withinsaid immunogenic polypeptide sequence. The location of such sequenceswithin the sequence of a polypeptide forming an immunogen of theinvention may affect relative immunogenic activity. In addition,immunogens of the present invention may comprise more than one proteincomprising the amino acid sequences disclosed herein. Such polypeptidesmay be part of a single composition or may themselves be covalently ornon-covalently linked to each other.

The immunogenic peptides disclosed herein may also be linked directlyto, or through a spacer or linker to: an immunogenic carrier such asserum albumin, tetanus toxoid, keyhole limpet hemocyanin, dextran, or arecombinant virus particle; an immunogenic peptide known to stimulate aT helper cell type immune response; a cytokine such as interferon gammaor GMCSF; a targeting agent such as an antibody or receptor ligand; astabilizing agent such as a lipid; or a conjugate of a plurality ofepitopes to a branched lysine core structure, such as the so-called“multiple antigenic peptide” described in (Posneft, D. N. et al., J.Biol. Chem., 263:1719–1725, (1988)); a compound such as polyethyleneglycol to increase the half life of the peptide; or additional aminoacids such as a leader or secretory sequence, or a sequence employed forthe purification of the mature sequence. Spacers and linkers aretypically comprised of relatively small, neutral molecules, such asamino acids and which are substantially uncharged under physiologicalconditions. Such spacers are typically selected from the group ofnonpolar or neutral polar amino acids, such as glycine, alanine, serineand other similar amino acids. Such optional spacers or linkers need notbe comprised of the same residues and thus may be either homo- orhetero-oligomers. When present, such linkers will commonly be of lengthat least one or two, commonly 3, 4, 5, 6, and possibly as much as 10 oreven up to 20 residues (in the case of amino acids). In addition, suchlinkers need not be composed of amino acids but any oligomericstructures will do as well so long as they provide the correct spacingso as to optimize the desired level of immunogenic activity of theimmunogens of the present invention. The immunogen may therefore takeany form that is capable of eliciting a CTL response.

In addition, the immunogenic peptides of the present invention may bepart of an immunogenic structure via attachments other than conventionalpeptide bonds. Thus, any manner of attaching the peptides of theinvention to an immunogen of the invention, such as an immunogenicpolypeptide as disclosed herein, could provide an immunogenic structureas claimed herein. Thus, immunogens, such as proteins of the invention,are structures that contain the peptides disclosed according to thepresent invention but such immunogenic peptides may not necessarily beattached thereto by the conventional means of using ordinary peptidebounds. The immunogens of the present invention simply contain suchpeptides as part of their makeup, but how such peptides are to becombined to form the final immunogen is left to the talent andimagination of the user and is in no way restricted or limited by thedisclosure contained herein.

The peptides that are naturally processed and bound to a class I MHCmolecule, and which are recognized by a tumor-specific CTL, need not bethe optimal peptides for stimulating a CTL response. See, for example,(Parkhurst, M. R. et al., J. Immunol., 157:2539–2548, (1996); Rosenberg,S. A. et al., Nat. Med., 4:321–327, (1998)). Thus, there can be utilityin modifying a peptide, such that it more readily induces a CTLresponse. Generally, peptides may be modified at two types of positions.The peptides may be modified at amino acid residues that are predictedto interact with the class I MHC molecule, in which case the goal is tocreate a peptide that has a higher affinity for the class I MHC moleculethan does the parent peptide. The peptides can also be modified at aminoacid residues that are predicted to interact with the T cell receptor onthe CTL, in which case the goal is to create a peptide that has a higheraffinity for the T cell receptor than does the parent peptide. Both ofthese types of modifications can result in a variant peptide that isrelated to a parent peptide, but which is better able to induce a CTLresponse than is the parent peptide. As used herein, the term “parentpeptide” means an oligopeptide with the amino acid sequence of SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or20.

The parent peptides disclosed herein can be modified by the substitutionof one or more residues at different, possibly selective, sites withinthe peptide chain. Such substitutions may be of a conservative nature,for example, where one amino acid is replaced by an amino acid ofsimilar structure and characteristics, such as where a hydrophobic aminoacid is replaced by another hydrophobic amino acid. Even moreconservative would be replacement of amino acids of the same or similarsize and chemical nature, such as where leucine is replaced byisoleucine. In studies of sequence variations in families of naturallyoccurring homologous proteins, certain amino acid substitutions are moreoften tolerated than others, and these are often show correlation withsimilarities in size, charge, polarity, and hydrophobicity between theoriginal amino acid and its replacement, and such is the basis fordefining “conservative substitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1—small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2—polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3—polar,positively charged residues (His, Arg, Lys); Group 4—large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 4—large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly nonconservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such radical substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,amino acids possessing non-standard R groups (i.e., R groups other thanthose found in the common 20 amino acids of natural proteins) may alsobe used for substitution purposes to produce immunogens and immunogenicpolypeptides according to the present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsyngeneic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would simultaneously be substituted.

Based on cytotoxicity assays, an epitope is considered substantiallyidentical to the reference peptide if it has at least 10% of theantigenic activity of the reference peptide as defined by the ability ofthe substituted peptide to reconstitute the epitope recognized by a CTLin comparison to the reference peptide. Thus, when comparing the lyticactivity in the linear portion of the effector:target curves withequimolar concentrations of the reference and substituted peptides, theobserved percent specific killing of the target cells incubated with thesubstituted peptide should be equal to that of the reference peptide atan effector:target ratio that is no greater than 10-fold above thereference peptide effector:target ratio at which the comparison is beingmade.

Preferably, when the CTLs specific for a peptide of SEQ ID NOs:1–20 aretested against the substituted peptides, the peptide concentration atwhich the substituted peptides achieve half the maximal increase inlysis relative to background is no more than about 1 mM, preferably nomore than about 1 μM, more preferably no more than about 1 nM, and stillmore preferably no more than about 100 pM, and most preferably no morethan about 10 pM. It is also preferred that the substituted peptide berecognized by CTLs from more than one individual, at least two, and morepreferably three individuals.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than 4 residues from thereference peptide, as long as they have substantially identicalantigenic activity.

It should be appreciated that an immunogen consisting only of a peptideof SEQ ID NO:1, or of a polypeptide that does not correspond to aprotein of SEQ ID NO:2, but nonetheless comprises a peptide of SEQ IDNO:1, or of a plurality of peptides of SEQ ID NOs:1–16 for HLA-a1/a11 orSEQ ID NO: 17–20 for HLA-A2, or of a polynucleotide coding for aplurality of peptides of SEQ ID NOs:1–20 is necessarily limited tostimulating a CTL response that is specific for those peptides found inthe immunogen. The immunogens of the invention can also comprise apolypeptide that itself comprises one or more of the epitopic peptidesof SEQ ID NOS: 1–20.

The immunogenic peptides and polypeptides of the invention can beprepared synthetically, by recombinant DNA technology, or they can beisolated from natural sources such as tumor cells expressing the parentprotein product.

The polypeptides and oligopeptides disclosed herein can be synthesizedin solution or on a solid support in accordance with conventionaltechniques. Various automated peptide synthesizers are commerciallyavailable and can be used in accordance with known protocols. See, forexample, (Grant, G. A., Synthetic Peptides: A User's Guide, 1992, W.H.Freeman and Company, New York; Coligan, J. E. et al, Current Protocolsin Protein Science, 1999, John Wiley & Sons, Inc., New York). Fragmentsof polypeptides of the invention can also be synthesized asintermediates in the synthesis of a larger polypeptide.

Recombinant DNA technology may be employed wherein a nucleotide sequencewhich encodes an immunogenic peptide or polypeptide of interest isinserted into an expression vector, transformed or transfected into anappropriate host cell, and cultivated under conditions suitable forexpression. These procedures are well known in the art to the skilledartisan, as described in (Coligan, J. E. et al, Current Protocols inImmunology, 1999, John Wiley & Sons, Inc., New York; Ausubel, F. M. etal, Current Protocols in Molecular Biology, 1999, John Wiley & Sons,Inc., New York; Molecular Cloning: A Laboratory Manual, 1989, ColdSpring Harbor Laboratory Press, Cold Spring Harbor). Thus, recombinantlyproduced peptides or polypeptides can be used as the immunogens of theinvention.

The coding sequences for peptides of the length contemplated herein canbe synthesized on commercially available automated DNA synthesizersusing protocols that are well know in the art. See for example, (Grant,G. A., Synthetic Peptides: A User's Guide, 1992, W.H. Freeman andCompany, New York; Coligan, J. E. et al, Current Protocols in ProteinScience, 1999, John Wiley & Sons, Inc., New York). The coding sequencescan also be modified such that a peptide or polypeptide will be producedthat incorporates a desired amino acid substitution. The coding sequencecan be provided with appropriate linkers, be ligated into suitableexpression vectors that are commonly available in the art, and theresulting DNA or RNA molecule can be transformed or transfected intosuitable hosts to produce the desired fusion protein. A number of suchvectors and suitable host systems are available, and their selection isleft to the skilled artisan. For expression of the fusion proteins, thecoding sequence will be provided with operably linked start and stopcodons, promoter and terminator regions, and a replication system toprovide an expression vector for expression in the desired host cell.For example, promoter sequences compatible with bacterial hosts areprovided in plasmids containing convenient restriction sites forinsertion of the desired coding sequence. The resulting expressionvectors are transformed into suitable bacterial hosts. Of course, yeast,insect, and mammalian host cells may also be used, employing suitablevectors and control sequences.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the genes of the present invention. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Ausubel, F. M. et al, CurrentProtocols in Molecular Biology, 1999, John Wiley & Sons, Inc., New York;Molecular Cloning: A Laboratory Manual, 1989, Cold Spring HarborLaboratory Press, Cold Spring Harbor). Such cells can routinely beutilized for assaying CTL activity by having said geneticallyengineered, or recombinant, host cells express the immunogenic peptidesof the present invention.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnon-transcribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

The polypeptide can be recovered and purified from recombinant cellcultures by methods including ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. High performance liquidchromatography (HPLC) can be employed for final purification steps.

The immunogenic peptides of the present invention may be used to elicitCTLs ex vivo from either healthy individuals or from cancer patientswith cancer, such as colorectal carcinoma, lung carcinoma, ovariancarcinoma, or prostate carcinoma. Such responses are induced byincubating in tissue culture the individual's CTL precursor lymphocytestogether with a source of antigen presenting cells and the appropriateimmunogenic peptide. Examples of suitable antigen presenting cellsinclude dendritic cells, macrophages, and activated B cells. Typically,the peptide at concentrations between 10 and 40μg/ml, would bepre-incubated with the antigen presenting cells for periods ranging from1 to 18 hrs. β₂-microglobulin (4 μg/ml) can be added during this timeperiod to enhance binding. The antigen presenting cells may also be heldat room temperature during the incubation period (Ljunggren, H.-G. etal., Nature, 346:476–480, (1990)) or pretreated with acid (Zeh, H. J.,III et al., Hum. Immunol., 39:79–86, (1994)) to promote the generationof denatured class I MHC molecules which can then bind the peptide. Theprecursor CTLs (responders) are then added to the antigen presentingcells to which the immunogenic peptide has bound (stimulators) atresponder to stimulator ratios of between 5:1 and 50:1, and mosttypically between 10:1 and 20:1. The co-cultivation of the cells iscarried out at 37° C. in RPMI 1640, 10% fetal bovine serum, 2 mML-glutamine, and IL-2 (5–20 Units/ml). Other cytokines, such as IL-1,IL-7, and IL-12 may also be added to the culture. Fresh IL-2-containingmedia is added to the cultures every 2–4 days, typically by removingone-half the old media and replenishing it with an equal volume of freshmedia. After 7–10 days, and every 7–10 days thereafter, the CTL arere-stimulated with antigen presenting cells to which immunogenic peptidehas been bound as described above. Fresh IL-2-containing media is addedto the cells throughout their culture as described above. Three to fourrounds of stimulation, and sometimes as many five to eight rounds ofstimulation, are required to generate a CTL response that can then bemeasured in vitro. The above described protocol is illustrative only andshould not be considered limiting. Many in vitro CTL stimulationprotocols have been described and the choice of which one to use is wellwithin the knowledge of the skilled artisan. The peptide-specific CTLcan be further expanded to large numbers by treatment with anti-CD3antibody. For example, see (Riddell, S. R. and Greenberg, P. D., J.Immunol. Methods, 128:189–201, (1990); Walter, E. A. et al., N. Engl. J.Med., 333:1038–1044, (1995)).

Antigen presenting cells that are to be used to stimulate a CTL responseare typically incubated with peptide of an optimal length, most commonlya nonapeptide, that allows for direct binding of the peptide to theclass I MHC molecule without additional processing. Larger oligopeptidesand polypeptides are generally ineffective in binding to class I MHCmolecules as they are not efficiently processed into an appropriatelysized peptide in the extracellular milieu. There a variety of approachesthat are known in the art, however, that allow oligopeptides andpolypeptides to be exogenously acquired by a cell, which then allows fortheir subsequent processing and presentation by a class I MHC molecule.Representative, but non-limiting examples of such approaches includeelectroporation of the molecules into the cell (Harding, C. H. III, Eur.J. Immunol., 22:1865–1869, (1992)), encapsulation of the molecules inliposomes which are fused to the cells of interest (Reddy, R. et al., J.Immunol. Methods, 141:157–163, (1991)), or osmotic shock in which themolecules are taken up via pinocytosis (Moore, M. W. et al., Cell,54:777–785, (1988)). Thus, oligopeptides and polypeptides that compriseone or more of the peptides of the invention can be provided to antigenpresenting cells in such a fashion that they are delivered to thecytoplasm of the cell, and are subsequently processed to allowpresentation of the peptides.

Antigen presenting cells suitable for stimulating an in vitro CTLresponse that is specific for one or more of the peptides of theinvention can also be prepared by introducing polynucleotide vectorsencoding the sequences into the cells. These polynucleotides can bedesigned such that they express only a single peptide of the invention,multiple peptides of the invention, or even a plurality of peptides ofthe invention. There are a variety of approaches that are known in theart, that allow polynucleotides to be introduced and expressed in acell, thus providing one or more peptides of the invention to the classI MHC molecule binding pathway. Representative, but non-limitingexamples of such approaches include the introduction of plasmid DNAthrough particle-mediated gene transfer or electroporation (Tuting, T.et al., J. Immunol., 160:1139–1147, (1998)), or the transduction ofcells with an adenovirus expressing the polynucleotide of interest(Perez-Diez, A. et al., Cancer Res., 58:5305–5309, (1998)). Thus,oligonucleotides that code for one or more of the peptides of theinvention can be provided to antigen presenting cells in such a fashionthat the peptides associate with class I MHC molecules and are presentedon the surface of the antigen presenting cell, and consequently areavailable to stimulate a CTL response.

By preparing the stimulator cells used to generate an in vitro CTLresponse in different ways, it is possible to control the peptidespecificity of CTL response. For example, the CTLs generated with aparticular peptide will necessarily be specific for that peptide.Likewise, CTLs that are generated with a polypeptide or polynucleotideexpressing or coding for particular peptides will be limited tospecificities that recognize those peptides. More broadly, stimulatorcells, and more specifically dendritic cells, can be incubated in thepresence of the whole protein. As a further alternative, stimulatorcells, and more specifically dendritic cells, can be transduced ortransfected with RNA or DNA comprising the polynucleotide sequenceencoding the protein. Under these alternative conditions, peptideepitopes that are naturally cleaved out of the protein, and which aregenerated in addition to peptide epitopes of SEQ ID NOs:1–20 canassociate with an appropriate class I MHC molecule, which may or may notinclude HLA-A1, -A2, or -A3. The selection of antigen presenting cellsand the type of antigen with which to stimulate the CTL, is left to theordinary skilled artisan.

In specific embodiments, the methods of the present invention include amethod for inducing a CTL response in vitro that is specific for a tumorcell expressing HLA-A1/A11 or HLA-A2, whereby the method comprisescontacting a CTL precursor lymphocyte with an antigen presenting cellthat has bound an immunogen comprising one or more of the peptidesdisclosed according to the invention.

In specific embodiments, the methods of the present invention include amethod for inducing a CTL response in vitro that is specific for a tumorcell expressing HLA-A1/A11 or HLA-A2, whereby the method comprisescontacting a CTL precursor lymphocyte with an antigen presenting cellthat has exogenously acquired an immunogenic oligopeptide or polypeptidethat comprises one or more of the peptides disclosed according to theinvention.

A yet additional embodiment of the present invention is directed to aprocess for inducing a CTL response in vitro that is specific for atumor cell expressing HLA-A1/A11 or HLA-A2, comprising contacting a CTLprecursor lymphocyte with an antigen presenting cell that is expressinga polynucleotide coding for a polypeptide of the invention and whereinsaid polynucleotide is operably linked to a promoter.

A variety of techniques exist for assaying the activity of CTL. Thesetechniques include the labeling of target cells with radionuclides suchas Na₂ ⁵¹Cr0₄ or ³H-thymidine, and measuring the release or retention ofthe radionuclides from the target cells as an index of cell death. Suchassays are well-known in the art and their selection is left to theskilled artisan. Alternatively, CTL are known to release a variety ofcytokines when they are stimulated by an appropriate target cell, suchas a tumor cell expressing the relevant class I MHC molecule and thecorresponding peptide. Non-limiting examples of such cytokines includeIFN-γ, TNFα, and GM-CSF. Assays for these cytokines are well known inthe art, and their selection is left to the skilled artisan. Methodologyfor measuring both target cell death and cytokine release as a measureof CTL reactivity are given in (Coligan, J. E. et al, Current Protocolsin Immunology, 1999, John Wiley & Sons, Inc., New York).

After expansion of the antigen-specific CTLs, the latter are thenadoptively transferred back into the patient, where they will destroytheir specific target cell. The utility of such adoptive transfer isdemonstrated in (North, R. J. et al., Infect. Immun., 67:2010–2012,(1999); Riddell, S. R. et al., Science, 257:238–241, (1992)). Indetermining the amount of cells to reinfuse, the skilled physician willbe guided by the total number of cells available, the activity of theCTL as measured in vitro, and the condition of the patient. Preferably,however, about 1×10⁶ to about 1×10¹², more preferably about 1×10⁸ toabout 1×10¹¹, and even more preferably, about 1×10⁹ to about 1×10¹⁰peptide-specific CTL are infused. Methodology for reinfusing the T cellsinto a patient are well known and exemplified in U.S. Pat. No. 4,844,893to Honski, et al., and U.S. Pat. No. 4,690,915 to Rosenberg.

The peptide-specific CTL can be purified from the stimulator cells priorto infusion into the patient. For example, monoclonal antibodiesdirected towards the cell surface protein CD8, present on CTL, can beused in conjunction with a variety of isolation techniques such asantibody panning, flow cytometric sorting, and magnetic bead separationto purify the peptide-specific CTL away from any remaining non-peptidespecific lymphocytes or from the stimulator cells. These methods arewell known in the art, and are their selection is left to the skilledartisan. It should be appreciated that generation of peptide-specificCTL in this manner, obviates the need for stimulating the CTL in thepresence of tumor. Thus, there is no chance of inadvertentlyreintroducing tumor cells into the patient.

Thus, one embodiment of the present invention relates to a process fortreating a subject with cancer characterized by tumor cells expressingcomplexes of HLA-A1/A11 or HLA-A2, whereby CTLs produced in vitroaccording to the present invention are administered in an amountsufficient to destroy the tumor cells through direct lysis or to effectthe destruction of the tumor cells indirectly through the elaboration ofcytokines.

Another embodiment of the present invention is directed to a process fortreating a subject with cancer characterized by tumor cells expressingany class I MHC molecule and an epitope of SEQ ID NO: 1–20, whereby theCTLs are produced in vitro and are specific for the epitope or parentprotein and are administered in an amount sufficient to destroy thetumor cells through direct lysis or to effect the destruction of thetumor cells indirectly through the elaboration of cytokines.

In the foregoing embodiments the cancer to be treated may include acolorectal carcinoma, an ovarian carcinoma, a lung carcinoma, andprostate carcinoma, but especially ovarian carcinoma.

The ex vivo generated CTL can be used to identify and isolate the T cellreceptor molecules specific for the peptide. The genes encoding thealpha and beta chains of the T cell receptor can be cloned into anexpression vector system and transferred and expressed in naïve T cellsfrom peripheral blood, T cells from lymph nodes, or T lymphocyteprogenitor cells from bone marrow. These T cells, which would then beexpressing a peptide-specific T cell receptor, would then haveanti-tumor reactivity and could be used in adoptive therapy of cancer,and more specifically cancer, colorectal carcinoma, ovarian carcinoma,lung carcinoma, and prostate carcinoma.

In addition to their use for therapeutic or prophylactic purposes, theimmunogenic peptides of the present invention are useful as screeningand diagnostic agents. Thus, the immunogenic peptides of the presentinvention, together with modern techniques of gene screening, make itpossible to screen patients for the presence of genes encoding suchpeptides on cells obtained by biopsy of tumors detected in suchpatients. The results of such screening may help determine the efficacyof proceeding with the regimen of treatment disclosed herein using theimmunogens of the present invention.

Alternatively, the immunogenic peptides disclosed herein, as well asfunctionally similar homologs thereof, may be used to screen a samplefor the presence of CTLs that specifically recognize the correspondingepitopes. The lymphocytes to be screened in this assay will normally beobtained from the peripheral blood, but lymphocytes can be obtained fromother sources, including lymph nodes, spleen, tumors, and pleural fluid.The peptides of the present invention may then be used as a diagnostictool to evaluate the efficacy of the immunotherapeutic treatmentsdisclosed herein. Thus, the in vitro generation of CTL as describedabove would be used to determine if patients are likely to respond tothe peptide in vivo. Similarly, the in vitro generation of CTL could bedone with samples of lymphocytes obtained from the patient before andafter treatment with the peptides. Successful generation of CTL in vivoshould then be recognized by a correspondingly easier ability togenerate peptide-specific CTL in vitro from lymphocytes obtainedfollowing treatment in comparison to those obtained before treatment.

The oligopeptides of the invention, such as SEQ ID NO: 1–20, can also beused to prepare class I MHC tetramers which can be used in conjunctionwith flow cytometry to quantitate the frequency of peptide-specific CTLthat are present in a sample of lymphocytes from an individual.Specifically, for example, class I MHC molecules comprising HLA-A2 andpeptides of SEQ ID NO: 1–16, or HLA-A1/A11 and peptides of SEQ IDNO:17–20, would be combined to form tetramers as exemplified in U.S.Pat. No. 5,635,363. Said tetramers would find use in monitoring thefrequency of CTLs specific for the combination of HLA-A1/A11 and, forexample, a peptide of SEQ ID NO:17 in the peripheral blood, lymph nodes,or tumor mass of an individual undergoing immunotherapy with thepeptides, proteins, or polynucleotides of the invention, and it would beexpected that successful immunization would lead to an increase in thefrequency of the peptide-specific CTL. Said tetramers could also bedeveloped for peptides of SEQ ID NOs:18–20 in combination withHLA-A1/A11. The same would hold true for uses of HLA-A2 and SEQ ID NO:1–16.

As stated above, a vaccine in accordance with the present invention mayinclude one or more of the hereinabove described polypeptides or activefragments thereof, or a composition, or pool, of immunogenic peptidesdisclosed herein. When employing more than one polypeptide or activefragment, such as two or more polypeptides and/or active fragments maybe used as a physical mixture or as a fusion of two or more polypeptidesor active fragments. The fusion fragment or fusion polypeptide may beproduced, for example, by recombinant techniques or by the use ofappropriate linkers for fusing previously prepared polypeptides oractive fragments.

The immunogenic molecules of the invention, including vaccinecompositions, may be utilized according to the present invention forpurposes of preventing, suppressing or treating diseases causing theexpression of the immunogenic peptides disclosed herein, such as wherethe antigen is being expressed by tumor cells. As used in accordancewith the present invention, the term “prevention” relates to a processof prophylaxis in which an animal, especially a mammal, and mostespecially a human, is exposed to an immunogen of the present inventionprior to the induction or onset of the disease process. This could bedone where an individual has a genetic pedigree indicating apredisposition toward occurrence of the disease condition to beprevented. For example, this might be true of an individual whoseancestors show a predisposition toward certain types of cancer.Alternatively, the immunogen could be administered to the generalpopulation as is frequently done for infectious diseases. Alternatively,the term “suppression” is often used to describe a condition wherein thedisease process has already begun but obvious symptoms of said conditionhave yet to be realized. Thus, the cells of an individual may havebecome cancerous but no outside signs of the disease have yet beenclinically recognized. In either case, the term prophylaxis can beapplied to encompass both prevention and suppression. Conversely, theterm “treatment” is often utilized to mean the clinical application ofagents to combat an already existing condition whose clinicalpresentation has already been realized in a patient. This would occurwhere an individual has already been diagnosed as having a tumor.

It is understood that the suitable dosage of an immunogen of the presentinvention will depend upon the age, sex, health, and weight of therecipient, the kind of concurrent treatment, if any, the frequency oftreatment, and the nature of the effect desired. However, the mostpreferred dosage can be tailored to the individual subject, asdetermined by the researcher or clinician. The total dose required forany given treatment will commonly be determined with respect to astandard reference dose as set by a manufacturer, such as is commonlydone with vaccines, such dose being administered either in a singletreatment or in a series of doses, the success of which will depend onthe production of a desired immunological result (i.e., successfulproduction of a CTL-mediated response to the antigen, which responsegives rise to the prevention and/or treatment desired). Thus, theoverall administration schedule must be considered in determining thesuccess of a course of treatment and not whether a single dose, given inisolation, would or would not produce the desired immunologicallytherapeutic result or effect.

The therapeutically effective amount of a composition containing one ormore of the immunogens of this invention, is an amount sufficient toinduce an effective CTL response to the antigen and to cure or arrestdisease progression. Thus, this dose will depend, among other things, onthe identity of the immunogens used, the nature of the diseasecondition, the severity of the disease condition, the extent of any needto prevent such a condition where it has not already been detected, themanner of administration dictated by the situation requiring suchadministration, the weight and state of health of the individualreceiving such administration, and the sound judgment of the clinicianor researcher. Thus, for purposes of prophylactic or therapeuticadministration, effective amounts would generally lie within the rangeof from 1.0 μg to about 5,000 μg of peptide for a 70 kg patient,followed by boosting dosages of from about 1.0 μg to about 1,000 μg ofpeptide pursuant to a boosting regimen over days, weeks or even months,depending on the recipient's response and as necessitated by subsequentmonitoring of CTL-mediated activity within the bloodstream. Of course,such dosages are to be considered only a general guide and, in a givensituation, may greatly exceed such suggested dosage regimens where theclinician believes that the recipient's condition warrants more aaggressive administration schedule. Needless to say, the efficacy ofadministering additional doses, and of increasing or decreasing theinterval, may be re-evaluated on a continuing basis, in view of therecipient's immunocompetence (for example, the level of CTL activitywith respect to tumor-associated or tumor-specific antigens).

For such purposes, the immunogenic compositions according to the presentinvention may be used against a disease condition such as cancer byadministration to an individual by a variety of routes. The compositionmay be administered parenterally or orally, and, if parenterally, eithersystemically or topically. Parenteral routes include subcutaneous,intravenous, intradermal, intramuscular, intraperitoneal, intranasal,transdermal, or buccal routes. One or more such routes may be employed.Parenteral administration can be, for example, by bolus injection or bygradual perfusion over time.

Generally, vaccines are prepared as injectables, in the form of aqueoussolutions or suspensions. Vaccines in an oil base are also well knownsuch as for inhaling. Solid forms which are dissolved or suspended priorto use may also be formulated. Pharmaceutical carriers, diluents andexcipients are generally added that are compatible with the activeingredients and acceptable for pharmaceutical use. Examples of suchcarriers include, but are not limited to, water, saline solutions,dextrose, or glycerol. Combinations of carriers may also be used. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques including sterile filtration. The resulting solutions may bepackaged for use as is, or the aqueous solutions may be lyophilized, thelyophilized preparation being combined with sterile water beforeadministration. Vaccine compositions may further incorporate additionalsubstances to stabilize pH, or to function as adjuvants, wefting agents,or emulsifying agents, which can serve to improve the effectiveness ofthe vaccine.

The concentration of the CTL stimulatory peptides of the invention inpharmaceutical formulations are subject to wide variation, includinganywhere from less than 0.01% by weight to as much as 50% or more.Factors such as volume and viscosity of the resulting composition mustalso be considered. The solvents, or diluents, used for suchcompositions include water, possibly PBS (phosphate buffered saline), orsaline itself, or other possible carriers or excipients.

The immunogens of the present invention may also be contained inartificially created structures such as liposomes, ISCOMS,slow-releasing particles, and other vehicles which increase theimmunogenicity and/or half-life of the peptides or polypeptides inserum. Liposomes include emulsions, foams, micelies, insolublemonolayers, liquid crystals, phospholipid dispersions, lamellar layersand the like. Liposomes for use in the invention are formed fromstandard vesicle-forming lipids which generally include neutral andnegatively charged phospholipids and a sterol, such as cholesterol. Theselection of lipids is generally determined by considerations such asliposome size and stability in the blood. A variety of methods areavailable for preparing liposomes as reviewed, for example, by (Coligan,J. E. et al, Current Protocols in Protein Science, 1999, John Wiley &Sons, Inc., New York)and see also U.S. Pat. Nos. 4,235,871, 4,501,728,4,837,028, and 5,019,369.

Liposomes containing the peptides or polypeptides of the invention canbe directed to the site of lymphoid cells where the liposomes thendeliver the selected immunogens directly to antigen presenting cells.Targeting can be achieved by incorporating additional molecules such asproteins or polysaccharides into the outer membranes of said structures,thus resulting in the delivery of the structures to particular areas ofthe body, or to particular cells within a given organ or tissue. Suchtargeting molecules may a molecule that binds to receptor on antigenpresenting cells. For example an antibody that binds to CD80 could beused to direct liposomes to dendritic cells.

The immunogens of the present invention may also be administered assolid compositions. Conventional nontoxic solid carriers includingpharmaceutical grades of mannitol, lactose, starch, magnesium,cellulose, glucose, sucrose, sodium saccharin, and the like. Such solidcompositions will often be administered orally, whereby apharmaceutically acceptable nontoxic composition is formed byincorporating the peptides and polypeptides of the invention with any ofthe carriers listed above. Generally, such compositions will contain10–95% active ingredient, and more preferably 25–75% active ingredient.

Aerosol administration is also an alternative, requiring only that theimmunogens be properly dispersed within the aerosol propellant. Typicalpercentages of the peptides or polypeptides of the invention are0.01%–20% by weight, preferably 1% –10%. The use of a surfactant toproperly disperse the immunogen may be required. Representativesurfactants include the esters or partial esters of fatty acidscontaining from 6 to 22 carbon atoms, such as caproic, octanoic, lauric,palmitic, stearic, linoleic, linolenic, olesteric and oleic acids withan aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters,such as mixed or natural glycerides may be employed. The surfactant mayconstitute 0.1–20% by weight of the composition, preferably 0.25–5%.Typical propellants for such administration may include esters andsimilar chemicals but are by no means limited to these. A carrier, suchas lecithin for intranasal delivery, may also be included.

The peptides and polypeptides of the invention may also be deliveredwith an adjuvant. Adjuvants include, but are not limited to complete orincomplete Freund's adjuvant, Montanide ISA-51, aluminum phosphate,aluminum hydroxide, alum, and saponin. Adjuvant effects can also beobtained by injecting a variety of cytokines along with the immunogensof the invention. These cytokines include, but are not limited to IL-1,IL-2, IL-7, IL-12, and GM-CSF.

The peptides and polypeptides of the invention can also be added toprofessional antigen presenting cells such as dendritic cells that havebeen prepared ex vivo. For example, the dendritic cells could beprepared from CD34 positive stem cells from the bone marrow, or theycould be prepared from CD14 positive monocytes obtained from theperipheral blood. The dendritic cells are generated ex vivo usingcytokines such as GM-CSF, IL-3, IL-4, TNF, and SCF. The cultured DC arethen pulsed with peptides at various concentrations using standardmethods that are well known in the art. The peptide-pulsed dendriticcells can then be administered either intraveneously, subcutaneously, orintradermally, and the immunization may also include cytokines such asIL-2 or IL-12.

The present invention is also directed to a vaccine in which animmunogen of the present invention is delivered or administered in theform of a polynucleotide encoding the a polypeptide or active fragmentas disclosed herein, whereby the peptide or polypeptide or activefragment is produced in vivo. The polynucleotide may be included in asuitable expression vector and combined with a pharmaceuticallyacceptable carrier. For example, the peptides or polypeptides could beexpressed in plasmid DNA and nonreplicative viral vectors such asvaccinia, fowipox, Venezuelan equine encephalitis virus, adenovirus, orother RNA or DNA viruses. These examples are meant to be illustrativeonly and should not be viewed as self-limiting A wide variety of othervectors are available and are apparent to those skilled in the art fromthe description given herein. In this approach, a portion of thenucleotide sequence of the viral vector is engineered to express thepeptides or polypeptides of the invention. Vaccinia vectors and methodsuseful in immunization protocols are described in U.S. Pat. No.4,722,848, the disclosure of which is incorporated herein by referencein its entirety.

Regardless of the nature of the composition given, additionaltherapeutic agents may also accompany the immunogens of the presentinvention. Thus, for purposes of treating tumors, compositionscontaining the immunogens disclosed herein may, in addition, containother antitumor pharmaceuticals. The use of such compositions withmultiple active ingredients is left to the discretion of the clinician.

In addition, the immunogens of the present invention can be used tostimulate the production of antibodies for use in passive immunotherapy,for use as diagnostic reagents, and for use as reagents in otherprocesses such as affinity chromatography.

The present invention also relates to antibodies that react withimmunogens, such as a polypeptide comprising one or more of the epitopicpeptides of SEQ ID NO: 1–20 as disclosed herein. Active fragments ofsuch antibodies are also specifically contemplated. Such antibodies, andactive fragments of such antibodies, for example, and Fab structure, mayreact with, including where it is highly selective or specific for, animmunogenic structure comprising 2, 3, 4 or more of the epitopicpeptides of the invention.

With the advent of methods of molecular biology and recombinanttechnology, it is now possible to produce antibody molecules byrecombinant means and thereby generate gene sequences that code forspecific amino acid sequences found in the polypeptide structure of theantibodies. Such antibodies can be produced by either cloning the genesequences encoding the polypeptide chains of said antibodies or bydirect synthesis of said polypeptide chains, with in vitro assembly ofthe synthesized chains to form active tetrameric (H₂L₂) structures withaffinity for specific epitopes and antigenic determinants. This haspermitted the ready production of antibodies having sequencescharacteristic of neutralizing antibodies from different species andsources.

Regardless of the source of the antibodies, or how they arerecombinantly constructed, or how they are synthesized, in vitro or invivo, using transgenic animals, such as cows, goats and sheep, usinglarge cell cultures of laboratory or commercial size, in bioreactors orby direct chemical synthesis employing no living organisms at any stageof the process, all antibodies have a similar overall 3 dimensionalstructure. This structure is often given as H₂L₂ and refers to the factthat antibodies commonly comprise 2 light (L) amino acid chains and 2heavy (H) amino acid chains. Both chains have regions capable ofinteracting with a structurally complementary antigenic target. Theregions interacting with the target are referred to as “variable” or “V”regions and are characterized by differences in amino acid sequence fromantibodies of different antigenic specificity.

The variable regions of either H or L chains contains the amino acidsequences capable of specifically binding to antigenic targets. Withinthese sequences are smaller sequences dubbed “hypervariable” because oftheir extreme variability between antibodies of differing specificity.Such hypervariable regions are also referred to as “complementaritydetermining regions” or “CDR” regions. These CDR regions account for thebasic specificity of the antibody for a particular antigenic determinantstructure.

The CDRs represent non-contiguous stretches of amino acids within thevariable regions but, regardless of species, the positional locations ofthese critical amino acid sequences within the variable heavy and lightchain regions have been found to have similar locations within the aminoacid sequences of the variable chains. The variable heavy and lightchains of all antibodies each have 3 CDR regions, each non-contiguouswith the others (termed L1, L2, L3, H1, H2, H3) for the respective light(L) and heavy (H) chains. The accepted CDR regions have been describedby Kabat et al, J. Biol. Chem. 252:6609–6616 (1977). The numberingscheme is shown in the figures, where the CDRs are underlined and thenumbers follow the Kabat scheme.

In all mammalian species, antibody polypeptides contain constant (i.e.,highly conserved) and variable regions, and, within the latter, thereare the CDRs and the so-called “framework regions” made up of amino acidsequences within the variable region of the heavy or light chain butoutside the CDRs.

The antibodies disclosed according to the invention may also be whollysynthetic, wherein the polypeptide chains of the antibodies aresynthesized and, possibly, optimized for binding to the polypeptidesdisclosed herein as being receptors. Such antibodies may be chimeric orhumanized antibodies and may be fully tetrameric in structure, or may bedimeric and comprise only a single heavy and a single light chain. Suchantibodies may also include fragments, such as Fab and F(ab₂)′fragments, capable of reacting with and binding to any of thepolypeptides disclosed herein as being receptors.

A further embodiment of the present invention relates to a method forinducing a CTL response in a subject comprising administering tosubjects that express HLA-A1 antigens an effective (i.e.,CTL-stimulating amount) of an immunogen of the invention that does notcomprise the entire protein expressing the epitopic peptides disclosedherein (i.e., one that comprises less than the entire protein where theprotein is a naturally occurring polypeptide) in an amount sufficient toinduce a CTL response to tumor cells expressing at least HLA-A1 orHLA-A2, as the case may be, thereby eliciting a cellular responseagainst said tumor cells.

A still further embodiment of the present invention relates to a methodfor inducing a CTL response in a subject, wherein the immunogen is inthe form of a polynucleotide. In one non-limiting example, the methodcomprises administering to subjects that express HLA-A1 at least one CTLepitope, wherein said epitope or epitopes are selected from a groupcomprising the peptides disclosed according to the invention, and arecoded within a polynucleotide sequence that does not comprise the entireprotein coding region, in an amount sufficient to induce a CTL responseto tumor cells expressing HLA-A1. A similar example could be drawn forHLA-A2.

While the below examples are provided to illustrate the invention, it isto be understood that these methods and examples in no way limit theinvention to the embodiments described herein and that other embodimentsand uses will no doubt suggest themselves to those skilled in the art.All publications, patents, and patent applications cited herein arehereby incorporated by reference, as are the references cited therein.It is also to be understood that throughout this disclosure where thesingular is used, the plural may be inferred and vice versa and use ofeither is not to be considered limiting.

EXAMPLE 1 Cell Lines

For HLA-A1/A11 studies, ARGOV57, a HLA-A1/11 positive ovarian cell line,was established by culturing tumor cells from an ascitic fluid from anovarian patient.

For HLA-A2 studies, OVCAR3, a HLA-A2 positive ovarian carcinoma cellline, was established by culturing tumor cells from an ascitic fluidfrom an ovarian patient.

SKOV3-A2, a HLA-A2 stably expressing ovarian carcinoma cell line, wasestablished by culturing tumor cells from an ascitic fluid from anovarian patient and transduced with HLA-A2 gene.

EXAMPLE 2 Immunoaffinity Purification

ARGOV57 cells were grown in 10-chamber Nunc cell factories (Fisher,Pittsburgh, Pa.). The cells were harvested by treatment with 0.45%trypsin and 0.32 mM EDTA, washed two times in phosphate-buffered salinesolution (pH 7.4), and stored as cell pellets at −80° C. Aliquots of6–8×10¹⁰ cells were solubilized at 5–10×10⁶ cells/ml in 20 mM Tris, pH8.0, 150 mM NaCl, 1% CHAPS, 18.5 μg/ml iodoacetamide, 5 μg/ml aprotonin,10 μg/ml leupeptin, 10 μg/ml pepstatin A, 5 mM EDTA, 0.2% sodium azide,and 17.4 μg/ml phenylmethylsulfonyl fluoride for 1 h. This and allsubsequent steps were performed with ice-cold solutions and at 4° C. Thelysates were then centrifuged at 100,000×g, the pellets discarded, andthe supernatants passed through a 0.22 μm filter. The supernatants werethen passed over a series of columns with the first containingSepharose, and the second containing the HLA-A1-specific monoclonalantibody, GAP-A1, bound to a protein A-Sepharose matrix. The secondcolumn was then sequentially washed with 20 column volumes of 20 mMTris, pH 8.0, 150 mM NaCl, 20 column volumes of 20 mM Tris, pH 8.0, 1.0M NaCl, and 20 column volumes of 20 mM Tris, pH 8.0. The peptides wereeluted from the column with 5 column volumes of 10% acetic acid. Theisolated HLA-A1 molecules were then boiled for 5 min to furtherdissociate any bound peptide from the heavy chains. The peptides werethen separated from the co-purifying class I heavy chain andβ₂-microglobulin by centrifugation on a Ultrafree-CL membrane with anominal molecular weight cut-off of 5,000 Daltons (Millipore, Beford,Mass.).

For a separate study, OVCAR3 or SKOV3 cells were successfully preparedusing the same procedure as just described except that A2 molecules wereprepared using A2 specific antibodies.

EXAMPLE 3 Peptide Fractionation

The peptide extracts were fractionated by RP-HPLC (Reversed Phase -HighPerformance Liquid Chromatography) using an Applied Biosystems (ABI)model 140B system. The extracts were concentrated by vacuumcentrifugation from about 20 ml down to 250 μl and injected into eithera Brownlee (Norwalk, Conn.) C₁₈ Aquapore column (2.1 mm×3 cm; 300 Å; 7μm) or a Higgins (Mountain View, Calif.) C18 Haisil column (2.1 mm×4 cm;300 Å; 5 μm). The peptides were eluted by first using a gradient ofacetonitrile/0.085% TFA (trifluoroacetic acid) in 0.1% TFA/water, withthe concentration of acetonitrile increasing from 0–9% (0–5 minutes),9–36% (5–55 minutes), and 36–60% (55–62 minutes). A second dimensionfractionation of combined fractions 17 and 18 from the first dimension(TFA) fraction was accomplished using the same gradient but with thesubstitution of HFBA (heptafluorobutyric acid) for TFA. The flow ratewas 200 μl/min, and fractions were collected at 1 min (Brownlee column)or 40 second (Higgins column) intervals. A third dimension of RP-HPLCwas achieved using an Eldex (Napa, Calif.) MicroPro Pump, a homemade C₁₈microcapillary column, and an ABI model 785A UV absorbance detector. Thecolumn was prepared by packing a 27 cm bed of 10 μm C₁₈ particles in asection of 285 μm o.d./75 μm i.d. fused silica (Polymicro Technologies,Phoenix, Ariz.). Peptides in combined fractions 26 and 27 of the seconddimension fraction were loaded onto this column and eluted with agradient of acetonitrile/0.67% triethylamine acetate/water in 0.1%triethylamine acetate/water, with the concentration of acetonitrileincreasing from 0–60% in 40 minutes. The flow rate was about 300 nl/min,and fractions were collected into 25 μl of water every 30 s. In allRP-HPLC experiments, peptides were detected by monitoring UV absorbanceat 214 nm.

EXAMPLE 4 Mass Spectrometric Analysis

The second dimension HPLC fraction was analyzed using an affluentsplitter on the microcapillary HPLC column. In this experiment, thecolumn (360 μm o.d.×100 μm i.d. with a 25 cm C₁₈ bed) was butt connectedwith a zero dead volume tee (Valco, Houston, Tex.) to two pieces offused silica of different lengths (25 μm and 40 μm i.d.). Peptides wereeluted with a 34 min gradient of 0–60% acetonitrile. The 25 μm capillarydeposited one-fifth of the HPLC effluent into the wells of a microtitreplate for use in CTL epitope reconstitution assays, whereas theremaining four-fifths of the effluent was directed into the massspectrometer. Ions were formed by electrospray ionization, and massspectra were recorded by scanning between mass to charge ratios (m/z)300 and 1400 every 1.5 seconds. Peptide sequences were determined by CAD(collision-activated dissociation) tandem mass spectrometry as describedin the literature (Hunt, D. F. et al., Proc. Natl. Acad. Sci. U.S.A.,83:6233–6237, (1986)).

EXAMPLE 5 Peptide Synthesis

Peptides were synthesized using a Gilson (Madison, Wis.) AMS 422multiple peptide synthesizer. Ten μMol quantities were synthesized usingconventional FMOC amino acids, resins, and chemical techniques. Peptideswere purified by RP-HPLC using a 4.6 mm×100 mm POROS (PerseptiveBiosystems, Cambridge, Mass.) column and a 10 min, 0–60% acetonitrile in0.1% TFA gradient.

EXAMPLE 6 Identification of Peptides that Associates with HLA-A1/A11

To identify the antigens present on the surface of the ARGOV57 ovariancarcinoma tumor cell line, HLA-A1/A11 molecules were purified byimmunoaffinity chromatography from 8×10¹⁰ ARGOV57 tumor cells. Afteracid elution of the affinity column and dissociation of the classI/peptide complexes by boiling, the peptides were separated from theHLA-A1/A11heavy chains and β2-microglobulin by membrane filtration,concentrated, and then fractionated by RP-HPLC using TFA as the organicmodifier. Analysis of the fragmented masses obtained from the CADallowed the determination of the peptide sequence as MLKDIIKEY (SEQ IDNO: 17). CAD of a synthetic peptide corresponding to SEQ ID NO:17unequivocally identified the unknown as having the sequence of SEQ IDNO:17. A Genbank search revealed that a peptide of SEQ ID NO:17corresponds exactly to amino acid residues 305–313 of the hepatocellularcarcinoma associated protein JCL-1.

Other sequences (SEQ ID NOs: 18–20) were determined in the same way.Thus, analysis of the fragmented masses obtained from the CAD allowedthe determination of the peptide sequence as TSYVKVLEH (SEQ ID NO: 18).CAD of a synthetic peptide corresponding to SEQ ID NO: 18 unequivocallyidentified the unknown as having the sequence of SEQ ID NO: 18. AGenbank search revealed that a peptide of SEQ ID NO: 18 correspondsexactly to amino acid residues 282–290 of the MAGE-4 protein. In thesame way, HEYLKAFKV (SEQ ID NO: 19) was shown to correspond exactly toresidues 152–160 of the milk fat globule-EGF factor 8 protein andIYIKQIKTF (SEQ ID NO: 20) corresponds exactly to a nonapeptide fromhuman retinoblastoma-associated protein.

EXAMPLE 7 Identification of Peptides that Associates with HLA-A2

To identify the antigens present on the surface of the OVCAR3 or SKOV3ovarian carcinoma tumor cell line, HLA-A2 molecules were purified byimmunoaffinity chromatography from 8×10¹⁰ OVCAR3 or SKOV3 tumor cells.After acid elution of the affinity column and dissociation of the classI/peptide complexes by boiling, the peptides were separated from theHLA-A2 heavy chains and β2-microglobulin by membrane filtration,concentrated, and then fractionated by RP-HPLC using TFA as the organicmodifier. Analysis of the fragmented masses obtained from the CADallowed the determination of the peptide sequence as KIMDQVQQA (SEQ IDNO: 1). CAD of a synthetic peptide corresponding to SEQ ID NO:1unequivocally identified the unknown as having the sequence of SEQ IDNO:1. A Genbank search revealed that a peptide of SEQ ID NO:1corresponds exactly to amino acid residues 1745–1753 of the human APCgene protein.

Other sequences (SEQ ID NOs: 2–16) were determined in the same way usingthe appropriate protein source and GenBank sequences. Thus, for example,analysis of the fragmented masses obtained from the CAD allowed thedetermination of the peptide sequence as RLQEDPPAGV (SEQ ID NO: 2). CADof a synthetic peptide corresponding to SEQ ID NO: 2 unequivocallyidentified the unknown as having the sequence of SEQ ID NO: 2. A Genbanksearch revealed that a peptide of SEQ ID NO: 2 corresponds exactly toamino acid residues of the human ubiquitin-conjugating enzyme.

TABLE 1 CTL-inducing peptide epitopes. SEQ ID NO: SEQUENCE SEQ ID NO:SEQUENCE 1 KIMDQVQQA 11 KLQELNYNL 2 RLQEDPPAGV 12 ILIEHLYGL 3 KLDVGNAEV13 YLIELIDRV 4 FLYDDNQRV 14 NLMEQPIKV 5 ALMEQQHYV 15 FLAEDALNTV 6YLMDTSGKV 16 TLLNVIKSV 7 ILDDIGHGV 17 MLKDIIKEY 8 LLDRFLATV 18 TSYVKVLEH9 LLIDDKGTIKL 19 HEYLKAFKV 10 RLYPWGVVEV 20 IYIKQIKTF

1. An isolated oligopeptide or peptide comprising at least one epitopicpeptide comprising the amino acid sequence of SEQ ID NO: 4, saidoligopeptide or peptide consisting of 9 to about 30 amino acid residues,wherein said oligopeptide or peptide binds to class I MHC molecules orcan be processed to bind to class I MHC molecules.
 2. The oligopeptideof claim 1 wherein said oligopeptide comprises at least two epitopicpeptides.
 3. The oligopeptide of claim 1 wherein said oligopeptidecomprises at least three epitopic peptides.
 4. An isolated oligopeptideor peptide comprising at least one epitopic peptide, said epitopicpeptide comprising one amino acid difference from SEQ ID NO: 4, saidoligopeptide or peptide consisting of 8 to about 30 amino acid residues,wherein said oligopeptide or peptide binds to class I MHC molecules orcan be processed to bind to class I MHC molecules.
 5. The oligopeptideor peptide of claim 4 wherein said one amino acid difference is theresult of a conservative amino acid substitution.
 6. The oligopeptide orpeptide of claim 4 wherein said one amino acid difference is thesubstitution of one hydrophobic amino acid with another hydrophobicamino acid.
 7. The oligopeptide or peptide of claim 4 wherein said aminoacid difference is the addition or deletion of one amino acid to or fromsaid epitopic peptide.
 8. A composition comprising an oligopeptide orpeptide of claim 1, 2, 3, 4, 5, 6, or 7 present in a pharmaceuticallyacceptable carrier and in an amount sufficient to elicit production ofantibodies or cells that react with said oligopeptide or peptide whensaid oligopeptide or peptide is administered to an immunologicallycompetent animal.
 9. An immunogen consisting of the amino acid sequenceof SEQ ID NO:
 4. 10. The oligopeptide of claim 2, said oligopeptidecomprising a first epitopic peptide and a second epitopic peptide,wherein said first epitopic peptide comprises the amino acid sequence ofSEQ ID NO: 4 and said second epitopic peptide comprises an amino acidsequence selected from the group consisting of SEQ ID NOS: 1–20.